The poxviridae comprise a large family of complex DNA viruses that replicate in the cytoplasm of vertebrate and invertebrate cells. The family of poxviridae can be divided into the subfamily chordopoxvirinae (vertebrate poxviruses) and entomopoxvirinae (insect poxviruses).
The chordopoxvirinae comprise several poxvirus species that can be used as vectors to express exogenous DNA segments encoding antigens against which an immune response is to be induced. Examples for poxviruses that can be used as live vaccines are Vaccinia virus and avipoxviruses, such as the canarypoxvirus and the fowlpoxvirus.
The use of Vaccinia viruses to engineer viral vectors for recombinant gene expression and for the potential use as recombinant live vaccines has been disclosed in numerous publications (see e.g. Mackett, M., Smith, G. L. and Moss, B. [1982] P.N.A.S. USA 79, 7415-7419; Smith, G. L., Mackett, M. and Moss, B. [1984] Biotechnology and Genetic Engineering Reviews 2, 383-407). To construct recombinant Vaccinia viruses, DNA sequences (genes), which code for foreign antigens are introduced into the genome of the Vaccinia virus under the regulation of suitable poxvirus promoters. If the gene is integrated at a site in the viral DNA, which is non-essential for the life cycle of the virus, the recombinant Vaccinia virus remains infectious. After infection the recombinant virus expresses the integrated DNA sequence (EP 83286 and EP 110385). The recombinant Vaccinia viruses prepared in this way can be used, on the one hand, as live vaccines for the prophylaxis of infectious diseases, and on the other hand, for the preparation of heterologous proteins in eukaryotic cells.
The use of Vaccinia virus as vector for the development of recombinant live vaccines has been affected by safety concerns and regulations. Most of the recombinant Vaccinia viruses described in the literature are based on the Western Reserve strain of Vaccinia virus. It is known that this strain has a high neurovirulence and is thus poorly suited for use in humans and animals (Morita et al., Vaccine 5, 65-70 [1987]). On the other hand the Modified Vaccinia virus Ankara (MVA) is known to be exceptionally safe. MVA has been generated by long-term serial passages of the Ankara strain of Vaccinia virus (CVA) on chicken embryo fibroblasts (for review see Mayr, A., Hochstein-Mintzel, V. and Stickl, H. [1975] Infection 3, 6-14; Swiss Patent No. 568392). MVA is distinguished by its great attenuation that is to say by diminished virulence or infectiosity while maintaining good immunogenicity. Recombinant MVA useful as vaccines have already been constructed and used in clinical trials. WO 98/13500 discloses a recombinant MVA containing and capable of expressing one or more DNA sequences encoding dengue virus antigens. The foreign DNA sequences were recombined into the viral DNA at the site of a naturally occurring deletion in the MVA genome.
Another approach towards the generation of safe and effective poxvirus vaccines utilizes avipoxviruses, e.g. canarypoxvirus and fowlpoxvirus, to express antigens to induce an immune response (U.S. Pat. No. 6,340,462). Avipoxviruses are naturally host-restricted and productively replicate only in avian species and cells (Taylor et al., Vaccine 1995, 13: 539-549). If human cells are infected with an avipoxvirus, heterologous genes are expressed from the viral genome. However, the avipoxvirus does not replicate in the human cells and there is, thus, no risk that the human being is harmed by productive virus replication. Various recombinant avipoxviruses have been constructed that express e.g. lentiviral gene products (U.S. Pat. No. 5,766,598), cytokines and/or tumor-associated antigens (U.S. Pat. No. 5,833,975) or rabies G glycoprotein (Taylor et al., Vaccine 1995, 13: 539-549). A recombinant canarypox virus expressing the four HIV genes gag, pol, env and nef has already been used in clinical trials (Peters, B. S., Vaccine 2002, 20: 688-705).
Since avipoxviruses productively replicate only in avian cells, these cells have to be used for the amplification of the virus and for the generation of recombinant viruses. Unfortunately, the titers of avipoxviruses obtained with avian cells are relatively low when compared to other poxviruses and it is, thus, more difficult to produce larger amounts of (recombinant) avipoxviruses in an industrial scale.